CXCL13 blockade disrupts B lymphocyte organization in tertiary lymphoid structures without altering B cell receptor bias or preventing diabetes in nonobese diabetic mice

Abstract

Lymphocytes that invade nonlymphoid tissues often organize into follicle-like structures known as tertiary lymphoid organs (TLOs). These structures resemble those found in spleen or lymph nodes, but their function is unknown. TLOs are recognized in many autoimmune diseases, including the NOD mouse model of type 1 diabetes. In some cases, TLOs have been associated with the B lymphocyte chemoattractant, CXCL13. Studies presented in this article show that CXCL13 is present in inflamed islets of NOD mice. Ab blockade of this chemokine unraveled B lymphocyte organization in islet TLOs, without reducing their proportion in the islets. These chaotic milieus contained B lymphocytes with the same distinct repertoire of B cell receptors as those found in mice with well-organized structures. Somatic hypermutation, associated with T-B interactions, was not impaired in these disorganized insulitis lesions. Finally, loss of B lymphocyte organization in islets did not provide disease protection. Thus, B lymphocytes infiltrating islets in NOD mice do not require the morphology of secondary lymphoid tissues to support their role in disease.

FIGURE 1

CXCL13 is expressed in inflamed islets of NOD mice. A, Gel electrophoresis showing RT-PCR products from isolated pancreatic islets using cxcl13-specific primers. First three lanes are from inflamed islets of three independent female NOD mice. Last three lanes are from islets of three independent female C57BL/6 mice. Lower lanes are controls using hprt primers from the same islets. B, Real-time PCR showing cxcl13 levels, normalized to hprt, from isolated islets. At least 20 islets were isolated from three mice per group, and pooled for each mouse. Normalized cxcl13 levels are significantly higher in islets from 12-wk-old NOD mice than from any other group. **p < 0.0001, compared with noninfiltrated NOR controls or 3-wk-old NOD mice; p < 0.001 for 6-wk-old NOD mice; p = 0.004 compared with 14-wk-old NOR mice. C, Immunohistochemical staining of frozen NOD pancreatic sections shows CXCL13⁺ (dark brown) cells among invading lymphocytes in early insulitis. Right panel shows control section processed with secondary reagents, but without primary anti-CXCL13 Ab (original magnification ×20).

FIGURE 2

CXCL13 receptor is expressed on B cells in pancreas and spleen. Flow cytometry shows CXCR5 expression on B220⁺ B cells from pancreas and spleen of the same female NOD mouse. Image is representative of data from six mice.

FIGURE 3

CXCL13 blockade does not stop B lymphocyte invasion of pancreatic islets, but scrambles tertiary lymphoid organization. A, Immunofluorescent staining of a typical, untreated, inflamed islet from a 12-wk-old female NOD mouse, with B220⁺ B cells (pseudocolored pink) surrounding CD3⁺ T cells (pseudocolored blue) (original magnification ×20). B, Immunofluorescent staining of a typical inflamed islet from a 12-wk-old female NOD mouse treated with CXCL13-blocking Ab from age 3 wk (original magnification ×20). C, Bar chart showing B lymphocytes as proportion of total lymphocytes extracted from pancreata of 12-wk-old female NOD mice treated with anti-CXCL13, isotype control, or not treated, analyzed by flow cytometry. Bar height shows average for three mice per group, with SD as indicated. D, Bar chart showing percent of inflamed islets classified as organized, disorganized, or intermediate from 12-wk-old female NOD mice treated with anti-CXCL13, isotype control Abs, or not treated. p < 0.001 for CXCL13 versus isotype-treated controls; p < 0.001 for CXCL13 versus untreated controls; p = 0.9 for isotype-treated controls versus untreated controls. Independent islets from anti-CXCL13, n = 34; isotype control, n = 27; untreated, n = 18; anti-CXCL13–treated mice, n = 3; isotype control mice, n = 2; untreated controls mice, n = 2.

FIGURE 4

CXCL13 blockade fails to alter selection of the B cell repertoire found in pancreatic islets. A, LC gene families expressed by B cells from pancreata (black bars) and draining PLNs (gray bars) of 12-wk-old prediabetic female V_H125/NOD mice treated with anti-CXCL13 from the age of 3 wk. p = 0.002 by Fisher exact test; mice, n = 3; independent clones, n = 53. B, The ratio of predominant B lymphocyte V_κ families found in pancreata, relative to those from PLNs, in untreated V_H125/NOD (black bars) and anti-CXCL13–treated V_H125/NOD mice (gray bars). p = 0.066; 95% CI 0.77–1.01 by Poisson regression; mice, n = 8; independent clones, n = 104.

FIGURE 5

CXCL13 blockade does not significantly decrease CDR mutations found in BCR LCs from pancreata. Gene segments encoding BCR κ LCs cloned from pancreata of V_H125/NOD mice were examined for evidence of somatic hypermutation as indicated by mutations in the CDRs. A, A total of 35% of BCR LC gene segments from pancreata of untreated controls have mutations in the CDR-encoding region. B, A total of 34% of BCR LC gene segments from anti-CXCL13–treated mice have mutations in the CDR-encoding regions. Keys show shading corresponding to number of mutations. Untreated, n = 45 clones; anti-CXCL13, n = 29 clones, from at least three mice per group. p = 0.861 by negative binomial regression.

FIGURE 6

CXCL13 blockade does not protect against development of diabetes. Female V_H125/NOD mice were administered monoclonal anti-CXCL13 (◆) or isotype control (■), or were untreated (▲). Blood glucose levels were checked weekly, and mice diagnosed with diabetes at >200 mg/dl. Study was stopped for lack of efficacy when at least 50% of anti-CXCL13–treated mice had become diabetic. Mice, n = 10 per group. Log rank p = 0.94 CXCL13 versus isotype control treated; log rank p = 0.27 anti-CXCL13 versus untreated controls.